PROJECT SUMMARY/ABSTRACT The increased prevalence of multi-drug resistant bacteria has led to an urgent unmet need to develop new an- timicrobial drugs. Targeting bacterial natural product biosynthesis has emerged as a promising avenue, due to the role of natural products as virulence factors, signaling factors, and agents of microbial warfare. However, to date, no drugs targeting enzymes in these biosynthetic pathways have reached the clinic. Hence, systematic investigations into key biosynthetic enzymes are necessary to understand fully their individual roles in virulence factor production. S-adenosyl-L-methionine (SAM)-dependent methyltransferases are ubiquitous throughout all domains of life and modify a diverse array of substrates, including proteins, nucleic acids, and natural products. In eukaryotes, methyltransferase activity modulates cellular processes such as epigenetic gene regulation and neuronal communication; consequently, these methyltransferases are extensively studied and have been validated as anticancer targets. In contrast, natural product methyltransferases have yet to be explored as therapeutic ?tar- gets?, in spite of their important role in the biosynthesis of pathogenic virulence factors and pharmaceutically relevant compounds. During biosynthesis, natural product methyltransferases recognize substrates that are covalently tethered to a carrier protein; hence, successful methyl transfer is a function of both substrate recog- nition and proper protein-protein interactions between the methyltransferase and the carrier protein. The tran- sient nature of these protein-protein interactions makes targeting methyltransferases particularly challenging, yet elucidating the molecular basis of cofactor-protein, substrate-protein, and protein-protein recognition would offer a significant step towards advancing these efforts. Herein, our overall goal is to identify and engineer critical methyltransferase interactions during the biosynthe- sis of non-ribosomal peptide and polyketide-derived virulence factors by combining organic synthesis, ad- vanced molecular biology, and structural biology. We propose to (aim 1) develop small molecules to interro- gate cofactor and substrate methyltransferase activity, (aim 2) design tools to capture methyltransferase-carrier protein interactions, and (aim 3) develop selective activity-based probes for bacterial natural product methyl- transferases for detection and identification in the bacterial proteome. The insights from this work will be broad- ly significant by providing critical first steps in structure-based design of drugs targeting bacterial methyltrans- ferases, fostering combinatorial biosynthetic efforts of alkyl groups into unnatural products with precise regio-, stereo-, and chemoselectivity, and advancing medicinal chemistry efforts that alter biological and physico- chemical properties of privileged pharmacophores through chemoenzymatic synthesis.